Colonic organoids and methods of making and using same

Abstract

Disclosed herein are methods for the in vitro differentiation of a precursor cell into definitive endoderm, which may further be differentiated into a human colonic organoid (HCO), via modulation of signaling pathways. Further disclosed are HCOs and methods of using HCOs, which may be used, for example, for the HCOs may be used to determine the efficacy and/or toxicity of a potential therapeutic agent for a disease selected from colitis, colon cancer, polyposis syndromes, and/or irritable bowel syndrome.

1 . A method of inducing formation of a human colon organoid (HCO), comprising the steps of
 a. contacting definitive endoderm (DE) with an FGF signaling pathway activator and a WNT signaling pathway activator for a period of time sufficient for said DE to form a mid-hindgut spheroid;   b. contacting the mid-hindgut spheroid of step (a) with a BMP activator and an EGF signaling pathway activator for a period of time sufficient to form said human colon organoid, wherein said human colon organoid expresses SATB2.   
     
     
         2 . The method of  claim 1  wherein said DE is derived from a precursor cell selected from an embryonic stem cell, and an induced pluripotent stem cell. 
     
     
         3 . The method of  claim 1 , wherein said FGF signaling pathway activator is selected from a small molecule FGF signaling pathway activator, a protein-based FGF signaling pathway activator, FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, or combinations thereof. 
     
     
         4 . The method of  claim 1 , wherein said WNT signaling pathway activator is selected from a protein Wnt signaling pathway activator, a small molecule Wnt signaling pathway activator, Lithium Chloride; 2-amino-4,6-disubstituted pyrimidine (hetero) arylpyrimidines; IQ1; QS11; NSC668036; DCA beta-catenin; 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl) pyrimidine, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, a GSK3 inhibitor, CHIR99021, or combinations thereof. 
     
     
         5 . The method of  claim 1 , wherein said BMP activator is selected from BMP2, BMP4, BMP7, BMP9, a small molecule that activates the BMP pathway, a protein that activate the BMP pathway, ventromorphins, and combinations thereof. 
     
     
         6 . The method of  claim 1 , wherein said period of time sufficient for said DE to form a mid-hindgut spheroid is determined by expression of CDX2 by said mid-hindgut spheroid of step (a). 
     
     
         7 . The method of  claim 1 , wherein said period of time sufficient for said mid-hindgut spheroid to form said human colon organoid by expression of SATB2 and CDX2 by a cell of said human colon organoid. 
     
     
         8 . The method of  claim 1 , wherein said HCO is characterized by the presence of colonic enteroendocrine cells (EEC). 
     
     
         9 . The method of  claim 1 , wherein said HCO is characterized by the presence of crypts and is substantially free of villi. 
     
     
         10 . The method of  claim 1 , wherein said HCO comprises colon-specific goblet cells. 
     
     
         11 . The method of  claim 1 , wherein said HCO is substantially free of Paneth cells. 
     
     
         12 . The method of  claim 1 , wherein said HCO secretes colon-specific hormone INSL5. 
     
     
         13 . An HCO obtained according to the method of  claim 1 . 
     
     
         14 . A method of forming colonic tissue, comprising engrafting the HCO of  claim 13 , under a kidney capsule of a mammal. 
     
     
         15 . A method of determining the efficacy and/or toxicity of a potential therapeutic agent for a disease selected from colitis, colon cancer, polyposis syndromes, and/or irritable bowel syndrome, comprising contacting said potential therapeutic agent with the HCO of  claim 13  for a period of time sufficient to determine the efficacy and/or toxicity of said potential therapeutic agent. 
     
     
         16 . An immunocompromised rodent comprising the HCO of  claim 13 . 
     
     
         17 . An intestinal colonoid derived from the HCO of  claim 13 . 
     
     
         18 . The intestinal colonoid of  claim 17 , wherein said intestinal colonoid is free of one or more of an immune function, innervation, blood vessels, villi, and Paneth cells.